Supernatant (100,000 x g, 45 min) fractions from cultures of normal dog kidney (MDCK-N) and cells transformed by Harvey murine sarcoma virus (MDCK-T) contained both cyclic AMP (cAMP) and cyclic GMP (cGMP) phosphodiesterase activities. MDCK-N cells exhibited much greater cAMP and cGMP phosphodiesterase activities (assayed at 0.5 muM substrate) than MDCK-T cells. MDCK-T cells contained some cGMP-stimulated cAMP phosphodiesterase activity which was not detected in supernatants from MDCK-N cells. Incubation of MDCK-T cells with sodium butyrate markedly increased cGMP-stimulated cAMP hydrolytic activities. This effect was not observed in MDCK-N cells. Maximal effects were observed after incubation of MDCK-T cells for 24 h with 1-2 mM sodium butyrate, concentrations known to induce differentiated cell functions in these cells and other cell lines. MDCK-N cells contain calcium- and calmodulin-stimulated phosphodiesterase activity. MDCK-T cells contain much less calmodulin-sensitive activity; incubation with butyrate did not restore this activity in MDCK-T cells. Thus, MDCK cells may provide a useful system to study the regulation of various types of cyclic nucleotide phosphodiesterase activities.